geosciences Article Survivability of Soil and Permafrost Microbial Communities after Irradiation with Accelerated Electrons under Simulated Martian and Open Space Conditions Vladimir Cheptsov 1,2,* ID , Elena Vorobyova 1,2, Andrey Belov 1, Anatoly Pavlov 3, Denis Tsurkov 3, Vladimir Lomasov 4 and Sergey Bulat 5,6 ID 1 Department of Soil Biology, Lomonosov Moscow State University, Moscow 119991, Russia; [email protected] (E.V.); [email protected] (A.B.) 2 Space Research Institute, Russian Academy of Sciences, Moscow 117997, Russia 3 Ioffe Physical-Technical Institute, Russian Academy of Sciences, St. Petersburg 194021, Russia; [email protected] (A.P.); [email protected] (D.T.) 4 Peter the Great St. Petersburg State Polytechnic University, St. Petersburg 194021, Russia; [email protected] 5 Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”, Gatchina 188300, Russia; [email protected] 6 Institute of Physics and Technology, Ural Federal University, Ekaterinburg 620002, Russia * Correspondence: [email protected]; Tel.: +7-929-917-07-80 Received: 1 July 2018; Accepted: 6 August 2018; Published: 8 August 2018 Abstract: One of the prior current astrobiological tasks is revealing the limits of microbial resistance to extraterrestrial conditions. Much attention is paid to ionizing radiation, since it can prevent the preservation and spread of life outside the Earth. The aim of this research was to study the impact of accelerated electrons (~1 MeV) as component of space radiation on microbial communities in their natural habitat—the arid soil and ancient permafrost, and also on the pure bacterial cultures that were isolated from these ecotopes. The irradiation was carried out at low pressure (~0.01 Torr) and low temperature (−130 ◦C) to simulate the conditions of Mars or outer space. High doses of 10 kGy and 100 kGy were used to assess the effect of dose accumulation in inactive and hypometabolic cells, depending on environmental conditions under long-term irradiation estimated on a geological time scale. It was shown that irradiation with accelerated electrons in the applied doses did not sterilize native samples from Earth extreme habitats. The data obtained suggests that viable Earth-like microorganisms can be preserved in the anabiotic state for at least 1.3 and 20 million years in the regolith of modern Mars in the shallow subsurface layer and at a 5 m depth, respectively. In addition, the results of the study indicate the possibility of maintaining terrestrial like life in the ice of Europa at a 10 cm depth for at least ~170 years or for at least 400 thousand years in open space within meteorites. It is established that bacteria in natural habitat has a much higher resistance to in situ irradiation with accelerated electrons when compared to their stability in pure isolated cultures. Thanks to the protective properties of the heterophase environment and the interaction between microbial populations even radiosensitive microorganisms as members of the native microbial communities are able to withstand very high doses of ionizing radiation. Keywords: astrobiology; Mars; accelerated electrons; gamma radiation; microbial communities; radioresistance; native environment; soil; permafrost Geosciences 2018, 8, 298; doi:10.3390/geosciences8080298 www.mdpi.com/journal/geosciences Geosciences 2018, 8, 298 2 of 24 1. Introduction According to current knowledge, life on Earth exists within a limited range of the balanced impacts of physical and chemical factors. Significant changes in the amplitude of the impact can lead to the disturbances of biosystems up to the disappearance of life. Despite the long-standing scientific interest in the problem of living cell resistance to adverse impacts, and numerous biological studies of extreme habitats, the question of the limits of cell viability in native environment under stress conditions remains open, and in recent decades has acquired the scale of an astrobiological problem. The viability of microorganisms under the influence of extraterrestrial cosmic factors is one of the priority issues of modern astrobiological studies [1–3]. Much attention is paid to ionizing radiation being considered as one of the main factors that can prevent the preservation and spread of life outside the Earth [4–10]. Indeed, among physicochemical factors that change the natural habitat and, above all, organisms per se, ionizing radiation is undoubtedly of prior importance having a direct effect on the genetic material of a living cell. The influence of ionizing radiation in relatively low doses on different organisms has been studied for a long time, and radiobiology generalizes and continues to accumulate experience of such studies [11–13]. Nevertheless, to plan and conduct astrobiological planetary studies, it is necessary to evaluate the effect of high doses of various components of cosmic radiation on microorganisms adapted to a native habitat [6–10]. In addition, the tasks of astrobiology require a differentiated approach to astrobiological models (Mars, ice satellites, Titan, etc.), taking into account hypothetical models of biospheres, as well as radiation fluxes and other data about the planet that characterize the environment. It has been proved that the conditions under which irradiation occurs (temperature, pressure, etc.) substantially correct radiation effects [5–7,10]. Microorganisms in natural heterophase mineral media (soils, sediments) are well protected against the stressful effects of physical and chemical factors. A lot of data there is published already on the increased resistance to stress of microorganisms in biofilms or microbial communities in situ in native habitat in comparison with pure microbial cultures [6,14–18]. Therefore, in order to correctly assess the radioresistance of terrestrial microorganisms in the extraterrestrial regolith or other habitat, the closest possible reproduction of the relevant physicochemical factors is necessary, while preserving the natural environmental model, to which microbial communities are adapted. Recent studies have shown that the resistance of microorganisms to gamma radiation is significantly underestimated [6–9,19]. Gamma radiation is a small part of cosmic radiation [5,10]. Different types of ionizing radiation differ in their biological effects [5]. For astrobiological modeling it is necessary to study in situ the stability of natural communities of microorganisms to various types of cosmic radiation in conditions that are as close as possible to extraterrestrial models. Elucidation of limiting doses and the identification of the most resistant in situ microorganisms or their communities will enable not only to correct the search tasks, but, despite the conventionality of extrapolating calculations to real conditions, it will allow for estimating the expediency of searching for life on a particular space object. The aim of this research was to study the effect of accelerated electrons as a component of cosmic radiation at high doses of 10 kGy and 100 kGy on microbial communities in their natural habitat—arid soil and ancient permafrost, as well as on pure bacterial cultures that were isolated from these biotopes. Irradiation was carried out under low pressure (~0.01 Torr) and low temperature (−130 ◦C) conditions, to simulate Martian and outer space environments. When considering the usage of different units of ionizing radiation dose in radiobiological literature we would clarify that gray (Gy) is defined as the absorption of one joule of radiation energy per kilogram of matter, and Gy is equal to 100 rad. A study of the effect of elevated radiation doses, which significantly exceed the amplitude fluctuations of the radiation on the surface of Mars [20–23], is necessary for the extrapolation of biological effects on the geological time scale. Laboratory experiments do not allow for studying the long accumulation of doses of relatively low intensity in the microbial biomass of natural soil. The Geosciences 2018, 8, 298 3 of 24 main question of the present study is how long the biosphere of Mars could be maintained after the supposed catastrophic change in planetary conditions [24–29], the gradual loss of the atmosphere [30], and the formation of a modern climate. 2. Materials and Methods 2.1. Objects of Study An arid soil sample (SN2) from the Negev desert and the ancient Arctic permafrost sample (M-1/91) were used as objects in the experiments, as well as pure cultures of bacteria Arthrobacter polychromogenes SN_T61 and Kocuria rosea SN_T60. The Arctic permafrost sample (M-1/91) was taken from the 1/91 well located in the Lower Kolyma Lowland (Yakutia) between the Bolshaya Chukochya and Malaya Konkovaja rivers in the sublatitudinal oler uplift [31]. The sample was taken at a depth of 34.0 m, from the ancient layers of the Oler suite, which did not melt for 1.8–2 million years. The temperature in the well was −7 ... −12 ◦C. Sampling and transportation methods and conditions were described earlier [32]. This sample was used earlier in our model experiments, with high doses of gamma irradiation at low temperature and low pressure [6,33]. The arid soil sample (SN2) was taken from the Negev desert in the Avdat (30◦47’ N/34◦46’ E) at a depth of 5–10 cm (horizon A) [34]. This area receives about 100 mm of precipitation during the whole year with months from June to October being the dry season with no precipitation at all. Soil-forming rocks are loess like loams. According to the WRB soil classification system, the soil is classified as Aridic Calcisols [35]. This sample was also used earlier in our model experiments with gamma irradiation at low temperature and low pressure [33]. The chemical characteristics of the samples are shown in Table1. Ion concentrations in aqueous extracts from the samples were determined using Dionex ICS-1100 Ion Chromatography System (Dionex Corporation, Sunnyvale, CA, USA), according to manufacturer recommendations; total organic carbon content was determined using the method of oxidation in potassium dichromate [36]. Table 1. Chemical characteristics of the samples studied.